Poster abstracts

Poster number 157 submitted by Lizette Zavala

Expanding the function and activity of a unique deadenylase involved in non-coding RNA processing and regulation

Lizette Zavala (Biochemistry and Molecular Biology, University of Chicago), Magadalena Sobien (Biochemistry and Molecular Biology, University of Chicago), Cassandra Hayne (Biochemistry and Molecular Biology, University of Chicago)

Abstract:
Target of the Egr1 (TOE1) is a unique 3'-to-5' Cajal-associated deadenylase involved in RNA processing of small non-coding RNAs (ncRNAs). It’s essential for trimming poly(A) tails for the proper maturation of small nuclear RNAs, and while it functions like other canonical deadenylases, TOE1 also has a unique 3'-exonuclease activity. TOE1 is an essential protein that is indispensable for early embryonic development of chordates, however, minimal biochemical studies characterization limits our understanding of the activity, stability, and regulation of this fascinating deadenylase1. Additionally, TOE1 has been linked to a neurological disorder—pontocerebellar hypoplasia type 7 (PCH7) due to the abundant accumulation of snRNA precursors upon TOE1 depletion in cells and the presence of specific point mutations in TOE12.
Here, we share our work to decipher how PCH7 linked TOE1 variants affect TOE1’s stability and activity by utilizing in vitro assays and interpret these findings in the context of AlphaFold generated models to provide mechanistic explanations for how these PCH7 linked mutations may cause disease. Using a recombinant system, we purified many TOE1 variants. Preliminary deadenylation assays and differential scanning fluorimetry (DSF) indicates distinct differences between wild-type TOE1 and PCH7-linked TOE1 variants. Our findings indicate that some variants impact TOE1’s thermal stability while others impact TOE1’s cleavage activity and possibly RNA recognition. We determined select mutations appear to have mild impacts on TOE1’s thermal stability from the DSF results, altered activity based on the deadenylation assays, and used AlphaFold models to decipher other ways mutations may impact TOE1 function or regulation. As an essential protein involved in the maturation of snRNAs, it is vital to discern the actual impact PCH7 TOE1 variants have, and to also expand our current understanding of how deadenylase can recognize and target their specific RNA substrates, deadenylate them, and aid in both protecting and moving the RNA substrates to their next stage in processing.

References:
1. Huynh, T. N., & Parker, R. (2023). The PARN, TOE1, and USB1 RNA deadenylases and their roles in non-coding RNA regulation. The Journal of biological chemistry, 299(9), 105139. https://doi.org/10.1016/j.jbc.2023.105139
2. Lardelli, R. M., et al. (2017). Biallelic mutations in the 3' exonuclease TOE1 cause pontocerebellar hypoplasia and uncover a role in snRNA processing. Nature genetics, 49(3), 457–464. https://doi.org/10.1038/ng.3762Skeparnias, I., Αnastasakis, D., Shaukat, A. N., Grafanaki, K., & Stathopoulos, C. (2017). Expanding the repertoire of deadenylases. RNA biology, 14(10), 1320–1325. https://doi.org/10.1080/15476286.2017.1300222

Keywords: RNA processing, Non-coding RNAs, deadenylase